Liposome immunoassay

ABSTRACT

A liposome or microsphere containing guaiac or other reagent is used in an immunoassay to detect an antigen or antibody. The guaiac or other reagent reacts with hemoglobin or other blood constituent to produce color indicating a positive result.

RELATED APPLICATIONS

This application is a continuation-in-part of our application Ser. No.08/951,214, filed Sep. 19, 1997 and now U.S. Pat. No. 5,919,633.

FIELD OF THE INVENTION

The invention herein disclosed is directed to immunoassays involvingliposomes or other microspheres.

BACKGROUND OF THE INVENTION

The basis for the invention is a modification of the Liposome ImmuneLysis Assay (LILA), whose theoretical principle is the following:

1. Prior Art Method

Antibody-dependent cytotoxicity is a process mediated by immunoglobulinsand a number of host proteins termed the “complement system”. Whenantibodies recognize and bind their target, they will sufferconformational changes that activate the complement system, which willeventually result in the formation of the “membrane attack complex” onthe surface of the foreign substance or cell. It is the membrane attackcomplex that perforates the cytoplasmic membrane and induces membranechannels that result in the lysis of the cell targeted by theantibodies. This perforation of membranes is utilized to evaluate thepresence of circulating antibodies. Instead of using cells or organisms,the LILA assay uses liposomes, which are small spheres composed oflipids. These liposomes have the property of allowing a substance placedin their interior to remain hidden from the environment as long as thelipid capsule or liposome remains intact. When the capsule is destroyedor punctured, its contents are released. The prior art disclosesliposomes that contain a substance which when released from the liposomewill change color when contacting an appropriate substrate present inthe surrounding environment. Further, liposomes may be sensitized withan antigen on their surface (i.e., cardiolipin). When the sensitizedliposomes are diluted with serum of a patient with antibodies to saidantigen, in this case against the syphilis antigen, i.e., cardiolipin,the antibodies recognize and attach to the surface of the sensitizedliposomes, thus activating the complement present in the serum, which inturn activates the membrane attack complex. Once the complement systemis activated and starts puncturing the liposome surface, the substancecontained in the interior of the liposome (e.g., an enzyme that maychange the color of a substrate) leaks into the surrounding solutionwhich contains the appropriate substrate for the enzyme. The color ofthe substrate changes due to the enzyme acting thereon. This colorchange can then be measured in a spectrophotometer or a device used forreading ELISA assays. This is an ingenious assay, but it does notprovide advantage over other methods such as solid phase immunoassayssuch as the ELISA, which are used to measure the same substance.

2. Prior Art

Tomioka et al in the Journal of Immunological Methods, Vol. 176 (1994)pages 1-7, teach immunoassay methods using liposomes using a model lipidof archaebacteria, namely,1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphlocholine. The advantageof this model lipid is that it could be used along with other lipids toform stable liposomes, as explained in the article.

Cole in U.S. Pat. No. 4,483,921 teaches immunoassays involvingliposomes. In that assay after recognition between antigen and antibody,the liposome ruptures releasing an enzyme which is detected indicating apositive results.

Wagner et al U.S. Pat. No. 4,978,625 uses liposomes in immunoassay,however, the assay does not depend upon the rupture of the liposome.

Gibbons et al (U.S. Pat. No. 5,068,198) describes preparing liposomes tobe used in immunoassays. The substance in the liposome is released, forexample, by freezing and thawing, sonication or osmotic shock.

Hosoda et al (U.S. Pat. No. 5,173,406) discloses a liposome immunoassayin which complement is involved in causing the rupture of liposome. Akit for carrying out the assay is taught. Various markers used in theassay are disclosed. Hosoda et al also disclose the use of polyclonal,as well as monoclonal antibodies.

Kida et al (U.S. Pat. No. 5,221,613) disclose an immunoassay processusing liposomes. The assay encapsulates an antibody in the liposome.

Malick et al (U.S. Pat. No. 5,620,903) teaches liposome assays.Water-insoluble dyes are used as the marker with water-soluble dyesadded as an option.

These prior art patents show liposomes used to encompass reagents invarious embodiments, however, none of the prior art reacts guaiacdisposed in a liposome with blood hemoglobin.

OBJECTS OF THE INVENTION

A main object of this invention is to produce a method that will allowfor the accurate and fast detection of a large number of human diseasesusing a very economical and simple one-step method.

A further object is to use sensitized liposomes or microspherescontaining a reagent to detect a blood component.

A specific object of this invention is to use a sensitized liposomecontaining guaiac to react with hemoglobin in the event a specificantibody is detected.

BRIEF DESCRIPTION OF THE INVENTION

The present disclosure describes an homogeneous immunoassay designed todetect the presence of specific antibodies in whole blood. Suchantibodies react with an antigen present on the surface of microcapsulessuch as liposomes, which enclose a reporter molecule. Subsequent to theantibody-antigen reaction, the microcapsules lyse by the action of thecomplement system, liberating their content which then reacts with asubstance or substances present in whole blood, producing a change incolor which can be visible to the naked eye.

SUMMARY OF THE INVENTION

The herein disclosed invention has a main object the rapid detection ofan antigen or antibody in the blood.

Another object is to produce an immunotest which can readily detect adisease state in the body by testing blood.

The invention contemplates a test kit for immunoassay of a blood samplecomprising in effective amounts:

a) an antigen or antibody sensitized liposome containing guaiac therein,

b) complement and

c) hydrogen peroxide

The invention also contemplates a method for detecting either an antigenor antibody in a hemoglobin-containing blood sample comprising

a) providing a container containing a sensitized liposome surroundingguaiac therein,

b) said container also being provided with complement and then

c) adding a hemoglobin containing blood sample to be tested for aspecific antigen or antibody such that if there is a reaction betweenthe antigen or antibody on the sensitized liposome, with its specificbinding partner, the liposome will rupture, releasing the guaiac toreact with the hemoglobin contained in the blood to produce ablue/brown-black color indicating a positive result; and in the eventthere is no reaction between the specific binding partners the liposomewill remain intact and the blood sample will retain its originalcoloration indicating a negative result. More specifically the liposomecan be sensitized to syphilis.

In a broad aspect, the described invention envisions in combination, acomposition to be tested comprising a blood sample and a sensitizedliposome containing a reagent which will react with a blood component.

In a broad aspect, the invention involves a method for detecting eitheran antigen or antibody in a blood sample comprising combining said bloodsample with a sensitized liposome containing a chemical agent which willreact with a blood component once a specific binding partner for thesensitized liposome causes the liposome to rupture.

This invention contemplates in combination, a composition to be testedcomprising a blood sample and a sensitized microsphere containing areagent which will react with a blood component, once the microsphere isruptured.

One aspect of this invention may be viewed as a method for detectingeither an antigen or antibody in a blood sample comprising combiningsaid blood with sensitized microspheres containing a chemical agentwhich will react with a blood component once a specific binding partnerfor the sensitized liposome causes the liposome to rupture.

Gibbons et al (U.S. Pat. No. 5,068,198) discloses materials forreversibly surrounding the reagents of this invention and are materialsthat are capable of temporarily confining the reagent until a reactionis effectuated and then releasing the confined reagent. Illustrativematerials include lipid bilayers such as liposomes, artificial cells,vesicles, natural cell membranes such as red blood cells ghosts; gelssuch as gelatin and agarose; polymerized beads and the like. Applicants'invention visualizes employing these microspheres or microcapsules intheir invention. Applicants also refer to U.S. Pat. No. 5,128,241 asemplifying “erythroctye ghost membrane” as an operative microcapsule touse in their invention. Note is also taken of Malick et al (U.S. Pat.No. 5,620,903) who disclose the use of silicon for forming microspheres.The disclosures of these patents are incorporated by reference into thedisclosure of this application.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D is a schematic representation of the reaction taking placebetween the liposome and blood sample to produce a color reaction.

FIGS. 2A-E is a schematic representation of the steps required to carryout the test.

FIG. 3 is a view illustrating a kit containing components necessary tocarry out the immunoassays of this invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIGS. 1A-D, there is illustrated a schematic representationof the immunoassay of this invention which takes place in the blood. Theimmunoassay involves the release of guiaiac 10 from a sensitizedliposome 12. In FIG. 1A guaiac 10 is disposed in a sensitized liposome12 which is sensitized with an antigen 16 (for example, syphilisantigen, etc.). The antigen sensitized liposome 12 is disposed withblood which is the media with which the immunoassay takes place. In theblood along with the antigen and sensitized liposome 12 are complement18 and antibodies 20. The antibody 20 recognizes its specific bindingpartner, namely, the antigen 16, on the liposome 12 and binds to theantigen 22 (FIG. 1B). Once there is binding between the antigen andantibody 22 on the liposome 12 in the presence of complement 18, theliposome 12 ruptures as at 24 releasing guaiac 10 (FIG. 1C) which, inturn, is oxidized by hemoglobin in blood in the presence of peroxidecausing the guaiac to turn blue 26 (FIG. 1D).

With reference to FIG. 2 A-E a lance 30 is used to obtain blood 32 fromthe finger 34 of a patient to be tested (FIG. 2A). The blood 32 dropsinto a test-tube 36 which has sensitized guaiac-containing liposomes 38at the bottom of the test-tube 36 (FIG. 2B). The blood 32 is mixed withguaiac-containing liposomes 38 (FIGS. 2 C-D). Referring to FIG. 2D,hydrogen peroxide 40 is added to the mixture of liposomes and blood 42with dropper 44. If the test is positive, a blue color 46 will form inthe bottom of the test-tube 36.

The method of this invention uses the same principle as theliposome/immunoassay of the prior art detailed above; however, themethod of this invention will embody test tubes containing the liposomesthat bear on their surfaces the antigen of interest. These liposomescontain in their interior guaiac or a derivative of guaiac which changescolor from transparent or light blue to dark blue when coming in contactwith hemoglobin and peroxide. The liposomes may be in a suspensioncontaining ammonium chloride or hypotonic saline or alternatively,normal saline (used to lyse the red cells and expose the hemoglobin) ina vacuum tube. The patient's blood will be drawn into such a tube.Without any further manipulation, the tube is allowed to stand at roomtemperature for 5-20 minutes (or alternatively at 37° C., to acceleratethe reaction). During this period of time the reaction takes place. Ifthe patient has the disease (that is, he carries antibodies against thespecific antigen sensitized to the antigen) the color of the liquid inthe tube containing the patient's blood will change from red to darkblue/brown-black. The mechanism of action is that if the patient hasantibodies to the specific antigen which sensitized the liposomes, theantibodies will bind to the surface of the liposomes and induce theircomplement mediated lysis, releasing the guaiac reagent. When the guaiacleaks from the liposomes, and reacts with the hemoglobin, a blue coloris produced in the presence of peroxide, indicating that the test waspositive. We envision the possibility of adding to the test tubeexogenous complement (such as guinea-pig serum) if the reaction requiresadditional complement source.

In one aspect, this invention involves peroxide and a liposomecontaining guaiac therein. This could be a commercial product sold tosuppliers for use in laboratories and doctors' offices. The liposomecontaining gulaiac could be sensitized with a specific antigen orantibody. The amount of guaiac in the liposome is not critical, butthere should be an amount adequate to show a color reaction withhemoglobin. The specific antigen or antigen may be syphilis. Theinvention embraces a test kit for immunoassay of a blood samplecomprising:

a) an antigen or antibody sensitized liposome containing guaiac therein,and

b) complement

The invention envisions a method for detecting either an antigen orantibody in a hemoglobin-containing blood sample comprising

a) providing a container containing a sensitized liposome surroundingguaiac therein,

b) said container also being provided with complement and peroxide andthen

c) adding a hemoglobin containing blood sample to be tested for aspecific antigen or antibody such that if there is a reaction betweenthe antigen or antibody on the sensitized liposome with its specificbinding partner, the liposome will rupture, releasing the guaiac toreact with the hemoglobin contained in the blood thus producing a bluecolor indicating a positive test; and in the event there is no reactionbetween the specific binding partners, the liposome will remain intactand the blood sample will retain its original coloration indicating anegative result.

Liposomes are small phospholipid bilayer vesicles that are capable ofcarrying lipophilic or hydrophilic molecules in their interior. As longas the vesicle is intact, its content is not in contact with theexterior milieu. If, however, the liposome's surface is perturbed, theinternal molecules are released and exposed to the external milieu andreact therewith to produce the assay result. The production of stableliposomes is well known to the art (see above mentioned references aswell as Schreier et al, U.S. Pat. No. 4,745,074.)

When attempting to detect the presence of a circulating antibody, theliposomes will bear on their surface the antigenic structures that arerecognized by such antibody. These antigens can either be incorporatedwith the liposome during their production (for example, by includingcardiolipin in the liposome composition for the detection of syphilisantibodies), or alternatively, they may be cross-linked to the surfaceof the liposomes as taught by Imai et al (U.S. Pat. No. 5,128,241); seealso Schreier et al (vide supra).

The lytic component of the blood will be mediated by the proteins of thecomplement system. When the antibodies present in the blood attach tothe antigen on the liposome surface, the complement system becomesactivated, resulting in the lysis of the liposomes, releasing the markersubstance to the exterior. A proportion of the liposomes may lack theinclusion of the marker in their interior, so as to reduce thenon-specific lysis of the marker-containing liposomes.

The marker molecule which will be present in the interior of theliposomes will be such that it can react with a substance present in thewhole unfractionated blood, and with or without the addition of anyfurther developer, will change the color of the whole reaction mixturein such a manner that it will be clearly visible to the naked eyewithout the need of special instrumentation or any further manipulation.

All required reagents are available commercially. The manufacture ofliposomes of predetermined specifications, with or without the presenceof internal markers and ligation of surface antigens, can be orderedfrom companies which provide these services. The antigens to be used canbe purchased commercially or manufactured following prior art.

EXAMPLE

Blood from a patient infected with syphilis will be obtained by thepuncture of the fingertip utilizing a commercially available lancet. Adrop (20 microliters) of said blood will be deposited in a small vial.Three drops of distilled water (which may be optional) will be added tothe vial in order to lyse the red cells, and subsequently 3 drops of aliposome mixture will be applied the liposomes in a concentration of 0.1micromoles of lipid per 100 microliters of reaction volume will be smallunilamellar vesicles including cardiolipin (syphilis antigen) as aconstituent, containing a saturated aqueous solution of guaiac gum(purchased form Sigma, St. Louis, Mo.) in their interior. Such liposomeswill be suspended in an isotonic solution of phosphate buffered salinecontaining thimerosal as preservative. The reaction mixture will beincubated for a period between 5 and 20 minutes at temperatures that mayrange between 22 and 37 degrees centigrade. At that point, a drop of 3%concentration of hydrogen peroxide in saline will be added, and inapproximately 30 seconds the whole mixture will be examined to note anychange in color from red (negative reaction) to dark brown/black(positive reaction, indicating that anti-cardiolipin antibodies werepresent in the blood).

An assay such as the above described will be available in the form of akit, so that 3 wells will be available for deposition of a drop of testblood. Well #1 will receive the reagents described above. Well #2 willbe the negative control, in which the liposomes will lack cardiolipin intheir structure, and a positive control in well #3, which will beidentical to #1, with the addition of one drop of a solution ofcommercially available goat anti-cardiolipin immunoglobulin at aconcentration of 1 milligram per milliliter. These positive and negativecontrols will allow the appropriate evaluation of the tested blood. Well#2 should remain red colored, and well #3 should turn dark brown/black.

This kit will have the following reagents:

1) Blood lancet.

2) A small tray with 3 wells with a total capacity of approximately 250microliters each.

3) A dropper bottle containing cardiolipin labeled liposomes with guaiacsolution in their interior mixed with an excess of empty unlabeledliposomes.

4) A dropper bottle with distilled water.

5) A dropper bottle with a mixture of unlabeled liposomes containing andlacking guaiac in their interior.

6) A dropper bottle with 3% hydrogen peroxide.

7) A dropper bottle with anti-cardiolipin immunoglobulins.

8) A small incubator that maintains the temperature of the reactionmixture at 37° C.

The principle of the assay is based on the use of lipid microspheresthat will be sensitive to attack by complement proteins assembled ontheir surface. It requires the use of very stable material, so that itdoes not leak out its contents spontaneously or leak as a result ofstorage aging. Although the technology of liposomes is relatively old,there are many products that have patents based on their improvement inperformance. For example, U.S. Pat. No. 5,620,903 to Malik et al teachesthe production of stabilized microspheres. There are many different waysto make liposomes or microspheres known in the art.

There are substances besides guaiac which will react with hemoglobin.Examples of these are chromogens that change color in the presence ofthe peroxidative activity of hemoglobin. These are Ortho tolidine,benzidine, Ortho dianisidine, diaminobenzidine, phenylenediamine andtetramethylbenzidine. All these methods require the presence of anoxygen donor in the form of hydrogen peroxide or hydroperoxide asdescribed in Pugia (U.S. Pat. No. 5,362,633) and a ferric ion. Otherindicator dyes such as phenothiazine, phenolphtalein and thiazolinonehydrazone among others will be operative.

The numerous kits for the detection of blood cells in urine utilizemostly benzidine derivatives. All the reagents are attached to the“dip-strip” which when dipped into the urine changes color in thepresence of red cells. The tests for occult blood in stool use eitherbenzidine derivatives, or guaiac. The latter is chosen because it haslower sensitivity and thus gives less false positives (in our case, evena minor leakage of reagents from the liposomes should not induce achange in color). As an indication of the sensitivity of guaiac tohemoglobin, there is a patent that teaches a VASELINE™ (Solidpetrolatum) based ointment containing guaiac and peroxides, so that whenperforming a rectal exam, the exploring finger glove will change colorif occult blood is present in the rectum. The prior art teaches theproduction of kits to detect red blood cells (occult blood) in bodyfluids.

Blood constituents other than hemoglobin are envisioned as being able toreact with reactants contained in the liposome or microspheres. Forexample, the invention envisions a chromogen in the liposome that reactswith protein. The liposomes could contain an appropriate solutionCoomassie blue, and if the blood to be tested has antibodies that inducecomplement to perforate the liposomes and release the Coomassie blue,this light orange-red substance would come in contact with the largeamount of proteins in the blood and change color to become darkblue/green, thus changing the color of the blood sample to blue/black.This would be a very simple assay, as there are large amounts ofproteins in the blood.

There are other substances in the blood which could potentially react tosome indicator in the liposomes. One could test for the activity ofvarious blood enzymes, for the presence of minerals and metals such asiron, and the list is very long. One could insert a colorant for iron inthe liposomes, and develop the reaction by the color produced by contactwith iron upon rupture of the liposomes/micro spheres.

The invention herein disclosed covers the detection of many diseases.Basically any infectious disease or autoimmune disease in which thesubject develops antibodies to a known blood antigen. For example, allinfectious diseases that are currently detected with the use ofimmunoassays such as ELISA or other such tests. These include: AIDS,Brucellosis, Tularemia, Plague, Glanders, Melioidosis, Anthrax, Typhoidfever, Tetanus, leptospirosis, syphilis, Rocky mountain spotted fever,pneumococccal pneumonia, menningococcemia (viral and bacterial),mycoplasma pneumonia, psittacosis, Coxsackievirus, echovirus,Parvovirus, Cytomegalovirus, measles, rubella, varicella, cat-scratchdisease, Lyme disease, ehrlichiosis, hepatis B, hepatitis C, HepatitisD, cryptococcosis, coccidioidomycosis, histoplasmosi, actinomycosis,nocardiosis, sleeping sickness, Chagas' disease, Leishmaniasis,Toxoplasmosis, Amebiasis and others.

Autoimmune diseases that could be diagnosed through this method include:Systemic Lupus Erythematosus, Rheumatoid Arthritis, Sjogren's Syndrome,Pemphigus, Bullous pemphigoid, and any other disease presentingcirculating autoantiboidies.

Any other disease in which a circulating substance can be detectedutilizing specific antibodies. For example, detection of circulatingcancer antigens by attaching specific antibodies to the surface of theliposomes. Or similarly, detection of viral particles (as in HIV) inblood. Moreover, this method may be utilized in the detection ofchemical agents, as well as pharmaceutical or illegal drugs in thecirculation.

The invention herein disclosed envisions the guaiac contained by theliposomes, and the hydroperoxide sitting by itself free in the testtube, or have the peroxide in the liposomes, and the guaiac free in thetube, or a mixture of some liposomes with guaiac and others withperoxide, or both in the same liposomes, or lastly, one of theingredients could be added from a dropper to the tube after the bloodextraction, however, this would add another step albeit a simple one.

One of the main ideas of this invention is to avoid using equipment thatreads optical densities or wave lengths, and develop a test that isrevealed clearly to the unaided eye. Yet there are ways to facilitatethe test. For example: one could have a small vial into which a drop ofblood obtained from a finger-stick is deposited as described in theexample. The drop of blood would change color as the reaction proceeds.The advantage of this would be that only one drop of blood needs to beobtained, thus avoiding the phlebotomy. This method is extensively usedin kits for home blood glucose measurements (actually now also used forhome AIDS test). Another advantage is that since there is no tube toopen, it is easy to add a drop of a reagent to a blood drop in a vial.In the event that one prefers a test in which a reagent or reagents areadded exogenously, this would probably allow for better control of theamounts of reagents to be utilized. A negative and positive control testvial will be included with the kit, to guarantee the lack of falsepositive or negative reactions.

Another instrument that would be of interest would be to have a warmingblock at 37 degrees to facilitate and accelerate the reaction. Thisshould not be very complicated or expensive, and could be provided withthe kit.

There are many advantages to this invention:

The test is an all or nothing response and is very easy to perform in asingle step.

It is self contained, requires no training or special equipment tointerpret results.

The test can be sold as a self standing kit, and would be particularlyuseful in settings that don't have highly specialized medical carefacilities. For some applications such as in AIDS, Syphilis, LupusErythematosus, Rheumatoid Arthritis, Hepatitis, Lyme's disease, RockyMountain Spotted Fever, as well as drug monitoring, this test couldbecome of routine use in laboratories and in physicians' offices.

The test is very rapid to perform. All that is necessary is to drawblood into a stored tube containing sensitized liposomes, complement,etc. and wait 5-20 minutes for test results.

The test is easy to read because there is a change of the color of theblood to blue/black positive (+), or the color does not change at allindicating negative (−).

The test does not require trained personnel. The nurse or technicianthat draws the blood is able to read the color change.

The test may be used for detecting numerous diseases: For example,infectious diseases of a known organism (AIDS, syphilis, hepatitis,etc.); autoimmune diseases with circulating antibodies (Lupus,Rheumatoid Arthritis, etc.)

The test does not require sending a blood sample to a central laboratoryfor analysis. So that not only is the test rapid, it is relativelyinexpensive and effects substantial cost savings in the health careindustry.

This invention allows for the first time a change of the color in wholeblood in an immunoassay; while all elements of blood are present, andnot having to separate the components or manipulate or treat the bloodsample prior to conducting the test.

The inventors have continued to make improvements to their invention.

Shown in FIG. 3 is an alternative kit 40 of components for carrying outthe immunoassay of this invention. The components are a test-tube 42containing therein sensitized liposomes 44. The sensitized liposomes cancontain therein guaiac, a selected dye to react with a blood componentor a peroxide such as hydrogen peroxide. The peroxide contained in thesensitized liposome is to be used in a special manner described herein.The kit will contain peroxide solution 46 when the sensitized liposomecontains guaiac; or contain guaiac when the liposome contains peroxidetherein. For special circumstances where the blood sample may notcontain adequate complement a container of complement in solution 47 isprovided. In addition, the kit contains a lancet 48 for obtaining theblood sample from the patient and a dropper 49 for use for applyingreagents to test-tube 42.

A further example of a kit is to be:

A Vacutainer™ (trademark of Becton-Dickinson) Tube containing

1) sensitized liposomes or liposomes containing the appropriate antigenon their surface with a chromogen (e.g. Guaiac) in the interior of theliposome and

2) Hydroperoxide (i.e. hydrogen peroxide)

The kit will be used in the following manner:

A blood sample is obtained by venipuncture into a Vacutainer™ tube.After lightly shaking, the tube will be maintained at 37 degreescentigrade or alternatively at room temperature. The antibodies bound tothe liposome surface will recognize its antigen and will mediate theactivation of complement and the subsequent lysis of the liposomesreleasing the chromogen to react with the hemoglobin in the presence ofHydroperoxide (hydrogen peroxide). Changing of the color of thechromogen indicates a positive test result.

In an embodiment of this invention, the blood sample to be used in thetest is obtained from a drop of blood obtained from a finger-stick,i.e., venipuncture into a Vacutainer™ tube. Enclosing all of thereactants in the tube as an “all in one” will allow the assay to beperformed in an enclosed tube with no need to manipulate the sample oradd reagent since all reagents will be enclosed in the single tube.

In a further alternative embodiment, there will also be a reagent in thetube which will mediate the lysis of the red cells (i.e., ammoniumchloride, water hypotonic buffers, etc.).

As an additive to the tube, there may be included an agent that willprevent the coagulation of the blood sample (i.e., citrate, heparin,etc.). It is to be understood that the anticoagulant component shouldnot interfere with any activation component such as complement, etc.

In an elegant embodiment of this intension, the Hydroperoxide (i.e.,hydrogen peroxide) is contained within the liposomes, and the chromogen(i.e., guaiac) is present freely in the mixture in the tube, that isexterior to the liposome. In other words, the reagent which getsliberated is the peroxide which facilitates the reaction between thechromogen and the hemoglobin.

The inventors envision encapsulating specific enzymes into the liposome.The enzymes in turn will react with a blood protein and be a componentin bringing about the reaction.

In an elegant embodiment of this invention, the chromogen is adhered tothe glass wall of the tube in the form of a plus sign (for example), andthe change in color of such area or sign may be visualized from theexterior of the glass tube once a positive reaction has taken place.

Other chromogens: Many substances can be incorporated in the liposomesso that upon their lysis, the released substance changes color due totheir recation with components of the blood. For example, Coomassie Bluecould be encapsulated, and upon release, will change to dark blue whenin contact with blood proteins. One skilled in the art could cite manyother substrates that could be acted upon by enzymes in the blood, orother chromogens that will change color when in contact with bloodsubstrates and blood elements.

An embodiment of this invention visualizes a variation of the test inwhich the liposome is not sensitized. In this embodiment, the inventorsutilize liposomes that contain the chromogen. However, the liposomeshave not been sensitized and do not contain an antigen on the liposomesurface. The antigen will be free in the tube. The activation ofcomplement in the mixture will be sufficient to induce the lysis of theliposomes, without necessitating their sensitization. This is based onthe principle of passive lysis, or bystander lysis, and is due to thelack of regulatory (control) proteins on the surface of the liposomes(contrary to what occurs on normal human cells).

Same as above but including substances that will prevent the complementinhibition by inactivating control proteins of the serum (such asanti-Factor I Fab '2).

Detection of circulating antigens (i.e., HIV, Hepatitis Virus, etc.) Theliposomes would not be sensitized. The Vacutainer™ tube will contain amixture that will include the presence of purified sheep or rabbitantibodies in the form of IgG or IgM against the antigen of interest(such as anti-HIV). The antigen will be recognized by the antibodies,and the formation of immune-complexes will activate the complementsystem, resulting in bystander lysis of liposomes containing chromogenor hydrogen peroxide (depending on the method of detection utilized).

Obviously, many modifications may be made without departing from thebasic spirit of the present invention. Accordingly, it will beappreciated by those skilled in the art that within the scope of theappended claims, the invention may be practiced other than has beenspecifically described herein.

We claim:
 1. A liposome containing guaiac therein.
 2. The liposome ofclaim 1 sensitized to cardiolipin.
 3. A method of detecting either anantigen or antibody in a hemoglobin-containing blood sample comprisinga) providing a container comprising an antigen or antibody-sensitizedliposome surrounding guaiac therein, said container further comprisingcomplement, and b) adding a blood sample which contains hemoglobin andwhich is to be tested for a specific antigen or antibody to saidcontainer, such that if the antigen or antibody on the sensitizedliposome contacts its specific antibody or antigen binding partner inthe blood sample, the liposome ruptures, releasing the guaiac therein toreact with the hemoglobin contained in the blood sample producing ablue/brown-black color as an indication of a positive test result and ifthe antigen or antibody on the sensitized liposome does not contact itsspecific antibody or antigen binding partner in the blood sample, theliposome remains intact and the blood sample retains its original coloras an indication of a negative test result.
 4. The method of claim 3wherein the liposome is sensitized to cardiolipin.
 5. In a method fordetecting either an antigen or antibody in a hemoglobin-containing bloodsample, the improvement comprising combining said hemoglobin-containingblood sample with a liposome sensitized by an antigen or antibody, saidliposome containing a combination of guaiac and hydroperoxide which willreact with hemoglobin once a specific binding partner for the sensitizedliposome in said blood sample causes the liposome to rupture allowingthe combination of guaiac and hydroperoxide to react with the hemoglobinthus indicating a positive test reaction.
 6. A method for detectingeither an antigen or antibody in a hemoglobin containing blood sample,said method comprising adding to an antigen or antibody sensitizedliposome containing peroxide therein, guaiac and a hemoglobin containingblood sample such that, if the antigen or antibody sensitized liposomecontaining peroxide therein contacts its binding partner in the bloodsample, the liposome will rupture releasing the peroxide to cause theguaiac to react with hemoglobin and change color thus indicating apositive test reaction.